The six-domain protein gelsolin functions in plasma (extracellular isoform) to disassemble actin filaments as part of the actin scavenging system. The D1 87N or Y mutation in domain 2 facilitates two aberrant proteolytic cleavages affording a 71 residue fragment that is found in amyloid fibrils in humans, putatively causing Familial Amyloidosis of Finnish type (FAF). We have discovered that the protease furin (found in the Golgi and endocytic compartments) and, potentially, related members of the prohormone convertase family effect the initial cleavage. This result allows us to probe the mechanism of this disease in an efficient fashion and provides a clear approach to intervene with small molecules. The second cleavage occurs at an unknown cellular/extracellular location. We have set out to understand the role of the FAF mutations in amyloidogenicity using biophysical studies in vitro (Aim 1) under conditions found in the trans-Golgi (pH 6.5, 200 uM Ca+2) in combination with cell biological studies focusing on both the export and import of gelsolin and fragments thereof in Aim 2, as well as transgenic animal studies in Aim 3. The goals of Aim 1 are to carry out a thermodynamic stability assessment on several different gelsolin constructs in addition to proteolysis sensitivity studies and structural comparisons of the WT and FAF variants to understand how these mutations lead to furin proteolysis. Small molecule inhibitors of the initial 172-173 cleavage will be sought using a cell-based screen. We will also carry out mechanistic and structural studies to better understand gelsolin fibril formation. In Aim 2, we will use a variety of biochemical, molecular and morphological approaches to further explore the site of processing of mutant gelsolin by furin and potentially other members of the prohormone convertase family. Moreover, we propose to identify the protease(s) involved in the second cleavage step leading to generation of the amyloidogenic fragment. In Aim 3, two transgenic animal models of FAF will be produced, one having one copy of WT and one copy of D187N plasma gelsolin (+/D187N) and the other having one copy of D187N on the knockout background (-/D187N) to improve our understanding of the relationship between aberrant processing, fibril formation and neurodegeneration. These mice provide a means of testing the small molecule therapeutics that come from Aims 1 and 2. Our long-term goal is to test the amyloid hypothesis in pathology by linking the cell biology and biophysical data to studies on transgenic animals and small molecule inhibitors that prevent misfolding and/or improper processing.